Project Summary Progressive heart failure is the leading cause of death worldwide. It is an epidemic with a survival rate of 50% over 5 years, affecting 6.5 million Americans. During a heart attack, a myocardial infarction (MI), the human heart loses 1 billion cardiomyocytes (CMs) on average (beating cells of the heart). Here, the heart?s inability to regenerate lost cardiomyocytes is well-known, leading to a significant decline in functional output as the once- healthy, contractile myocardium is now a scar tissue that does not contribute to the force production of a beating heart. Current treatment options are limited to palliative drug regimens (ACE inhibitors, beta blockers) or ventricular assist devices (risk of infection, thrombosis, power supply), and, the only real cure historically has been a heart transplant (limited supply). Thus, we have shown that a stem-cell based approach with stem-cell- derived-CMs for transplantation post-MI shows promise in regenerating the heart. These transplants form long- term grafts that can beat synchronously with host myocardium in mice, rats, and guinea pigs. Even moreso, we recently completed a 4-year pivotal study in macaque monkeys that revealed stem-cell-derived-CMs show nearly complete recovery of ejection fraction (the amount of blood pumped with each beat). However, despite this progress, there are still several outstanding limitations keeping stem-cell-derived-cardiomyocytes from being an effective therapy. Notably, single-cell-suspensions are the current delivery method to the heart making effective engraftment a challenge: <20% of injected cells persist as long-term, stable grafts, thus, lending to high manufacturing costs, and limiting the amount of new myocardium (heart muscle) that can form. Cell survival and retention could be significantly improved with the use of a biomaterial platform. In the past, biomaterial options for engineered heart tissues have been cardiac patches or cells sheets, but their geometries limit these constructs from electrically coupling with host myocardium and must be directly sutured onto the myocardium (more invasive). However, the use of an injectable biomaterial, such as a hydrogel that can gel in situ (directly mixed with cells), is appealing. They can be delivered directly through a catheter into myocardium, provide easy support and dispersion of transplanted cells directly at the site of MI, and provide a scaffold for the cells. Zwitterionic Injectable Pellet (ZIP) microgels are biodegradable, have easily tunable chemistry, and can be functionalized to support the needs of encapsulated CMs. In Aim 1, we will address the suitability for ZIP to aid in cell survival and retention in vitro, to discover optimal gel formulation (microgel size, cell attachment to gel), as well as cell survival and proliferation within the gel. In Aim 2, we will test the hypothesis that our ZIP gel can improve cardiac regeneration in vivo when used as an injectate for stem-cell- derived-CMs by evaluating both (1) cell survival, proliferation, and engraftment histologically, and, (2) overall functional outcome via MRI and echocardiography. Studies in this proposal will directly impact and challenge current delivery methods for stem-cell-derived-CMs transplanted for cardiac repair.